The proposed research seeks to determine the involvement of the DNA relaxing enzyme in simian virus 40 (SV40) and eukaryotic DNA replication. SV40 DNA replication can serve as a model for cellular DNA replication since, like cellular DNA, SV40 DNA is replicated in the nucleus by cellular enzymes and is associated with cellular histones to form the characteristic nucleosome structure. Using a low ionic strength isolation procedure, SV40 chromosomes isolated from infected monkey cells retain the ability to replicate in vitro when supplied with deoxy- and ribonucleoside triphosphates. Thus, enzymes which participate in in vivo DNA replication must be bound to the replicating chromosomes (RC). Preliminary studies show that the DNA relaxing enzyme co-sediments with RC in neural sucrose gradients and thus may be bound to SV40 chromsomes and participate in their replication. The DNA relaxing enzyme complex which co-sediments with RC will be further purified and characterized in order to demonstrate whether or not the enzyme is specifically bound to RC. A specific association with RC will be suggested if the enzyme is required for in vitro DNA synthesis after fractionation and reconstitution of RC. The enzyme will be purified and the degree of purity and molecular weight assessed by gel electrophoresis. The purified and complexed enzymes will be compared with respect to effects of inhibitors and effects of reagents which react with sulfhydryl groups.